Lipid formulations comprising a thiolated antioxidant

ABSTRACT

The present invention provides a formulation comprising a lipid matrix; at least one thiolated antioxidant; at least one bioactive agent; and optionally at least one chelating agent. The bioactive agent may be a gonadotrophin-releasing hormone (GnRH) agonist; a gonadotrophin-releasing hormone (GnRH) antagonist; a luthenizing hormone releasing hormone (LHRH); and/or a luthenizing hormone releasing hormone (LHRH) related peptide.

FIELD OF THE INVENTION

The present invention relates to lipid formulations and particularly tolipid formulations suitable for administration to subjects. Moreparticularly, the present invention relates to lipid compositions whichform non-lamellar phases and in which oxidative degradation is asignificant factor with regard to stability. Controlled-release lipidformulations are particularly suitable.

BACKGROUND TO THE INVENTION

Many bioactive agents including pharmaceuticals, nutrients, vitamins andso forth have a “functional window”. That is to say that there is arange of concentrations over which these agents can be observed toprovide some biological effect. Where the concentration in theappropriate part of the body (e.g. locally or as demonstrated by serumconcentration) falls below a certain level, no beneficial effect can beattributed to the agent. Similarly, there is generally an upperconcentration level above which no further benefit is derived byincreasing the concentration. In some cases increasing the concentrationabove a particular level results in undesirable or even dangerouseffects.

Some bioactive agents have a long biological half-life and/or a widefunctional window and thus may be administered occasionally, maintaininga functional biological concentration over a substantial period of time(e.g. 6 hours to several days). In other cases the rate of clearance ishigh and/or the functional window is narrow and thus to maintain abiological concentration within this window regular (or even continuous)doses of a small amount are required. This can be particularly difficultwhere non-oral routes of administration (e.g. parenteral administration)are desirable. Furthermore, in some circumstances, such as in thefitting of implants (e.g. joint replacements or oral implants) the areaof desired action may not remain accessible for repeated administration.In such cases a single administration must provide active agent at atherapeutic level over the whole period during which activity is needed.

Various methods have been used and proposed for the sustained release ofbiologically active agents. Such methods include slow-release, orallyadministered compositions, such as coated tablets, formulations designedfor gradual absorption, such as transdermal patches, and slow-releaseimplants such as “sticks” or minature syringe-type devices implantedunder the skin.

One method by which the gradual release of a bioactive agent has beenproposed is a so-called “depot” injection. In this method, a bioactiveagent is formulated with carriers providing a gradual release of activeagent over a period of a number of hours or days. These are often basedupon a degrading matrix which gradually disperses in the body to releasethe active agent.

The most common of the established methods of depot injection reliesupon a polymeric depot system. This is typically a biodegradable polymersuch poly (lactic acid) (PLA) and/or poly (lactic-co-glycolic acid)(PLGA) and may be in the form of a solution in an organic solvent, apre-polymer mixed with an initiator, encapsulated polymer particles orpolymer microspheres. The polymer or polymer particles entrap the activeagent and are gradually degraded releasing the agent by slow diffusionand/or as the matrix is absorbed. Examples of such systems include thosedescribed in U.S. Pat. Nos. 4,938,763, 5,480,656 and 6,113,943 and canresult in delivery of active agents over a period of up to severalmonths. These systems do, however, have a number of limitationsincluding the complexity of manufacturing and difficulty in sterilising(especially the microspheres). The local irritation caused by the lacticand/or glycolic acid which is released at the injection site is also anoticeable drawback. There is also often quite a complex procedure toprepare the injection dose from the powder precursor, and this proceduremust be conducted at the point of care just prior to administration.

From a drug delivery point of view, polymer depot compositions also havethe disadvantage of accepting only relatively low drug loads and havinga “burst/lag” release profile. The nature of the polymeric matrix,especially when applied as a solution or pre-polymer, causes an initialburst of drug release when the composition is first administered. Thisis followed by a period of low release, while the degradation of thematrix begins, followed finally by an increase in the release rate tothe desired sustained profile. This burst/lag release profile can causethe in vivo concentration of active agent to burst above the functionalwindow immediately following administration, and then drop back throughthe bottom of the functional window during the lag period beforereaching a sustained functional concentration. Evidently, from afunctional and toxicological point of view this burst/lag releaseprofile is undesirable and could be dangerous. It may also limit theequilibrium concentration which can be provided due to the danger ofadverse effects at the “peak” point.

A highly effective non-polymeric depot system was disclosed inWO2005/117830, in which a combination of a diacyl lipid or tocopherol, aphospholipid, and an oxygen containing organic solvent are combined toprovide a controlled-release matrix. Such a system has considerableadvantages, including a transition from low-viscosity to high-viscosityupon exposure to an aqueous environment, and the facility to provide agradual release of active agent over a long period from a biocompatibleand biodegradable composition. The disclosure of this document is herebyincorporated herein by reference.

Lipid-based systems such as that discussed above can also be used forother purposes by suitable choice of components, including the lipids,solvents and other additives used and their proportions. Such systemshave advantages in being able to solubilise and deliver certain activeagents which are otherwise difficult to dissolve for standardadministration methods (e.g. WO2005/046642). Furthermore, thecompositions can be selected to be bioadhesive, which allows delivery ofactive agents to a body surface over a sustained period (e.g.WO2006/075123).

The components of the delivery systems indicated above are highlybiotolerable. Indeed, many of these are endogenous lipids, and can bebeneficial even in the absence of an active pharmaceutical ingredient(API). This is particularly the case for bioadhesive formulations suchas those indicated above, where the skin or mucosal surface may besoothed and/or protected by the composition itself, aside from anyaction by any API.

One limitation of previously known lipid controlled-release formulationsis that many active agents and even the lipid components themselves canbe susceptible to oxidative degradation. Various antioxidant compoundsare known to offer some protection against this oxidative degradationbut few of these are compatible with lipid based systems. It wouldtherefore be of considerable advantage to provide an antioxidant/lipidcombination which was compatible and provided protection of an activeagent (such as an API) and/or of at least one lipid component againstoxidative degradation.

The present inventors have now surprisingly established thatthiol-containing antioxidants are unusually well suited to lipidformulations, and thus allow for such formulations to be stored forlonger periods and/or have longer duration of action than other types ofantioxidants previously tested.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention therefore provides aformulation comprising:

-   -   i) a lipid matrix;    -   ii) at least one thiolated antioxidant;    -   iii) optionally and preferably at least one bioactive agent; and    -   iv) optionally at least one chelating agent.

The lipid matrix preferably comprises at least one phospholipid, atleast one diacyl glycerol, at least one oxygenated organic solvent andoptionally at least one fragmentation agent, as described herein below.It is preferable that optional bioactive agent (iii) dissolves ordisperses in the remaining components to provide a low-viscositymixture.

To the inventors' knowledge, it has not previously been suggested thatthiolated antioxidants provide the surprising advantage in effectivenessin combination with lipid formulations.

In a further aspect, the present invention thus also provides a methodfor reducing oxidative degradation in a formulation comprising a lipidmatrix and at least one bioactive agent, said method comprising addingat least one thiolated antioxidant and optionally at least one chelatingagent. The various embodiments of this aspect are as described hereinwith respect to the formulation aspect.

In a yet further aspect, the present invention also provides the use ofa thiolated antioxidant in the reduction of oxidative degradation in aformulation comprising a lipid matrix and at least one bioactive agent.The various embodiments of this aspect are as described herein withrespect to the formulation aspect

DETAILED DESCRIPTION OF THE INVENTION

In all aspects, the present invention relates to the inclusion of athiolated antioxidant into a lipid matrix for the purpose of reducingoxidative degradation of at least one component of the overallformulation. Any lipid based matrix may be used in the invention, andthe effectiveness of the thiolated antioxidants may easily and routinelybe tested by methods described herein, with particular reference to theExamples.

Lipid matrices are of particular utility in bioadhesive formulations(with or without bioactive agents such as APIs) and/or incontrolled-release formulations whereby a bioactive agent (as describedherein, including APIs) is formulated with the lipid matrix andthiolated antioxidant and is delivered to a subject over an extendedperiod controlled by the nature of the lipid matrix and the phasebehaviour of the formulation. In all cases, it is an advantage for thelipid matrix as formulated according to the present invention to undergoa change of phase following administration. In particular, it ispreferable that the lipid matrix be in a low viscosity phase, such aslamellar or L₂ phase prior to administration and generate a non-lamellarphase, such as a liquid crystalline or L₃ phase after administration.Phase behaviour is further discussed below and applies to all aspects ofthe invention.

From a compositional point of view, it is an advantage if the lipidmatrices of the present invention are highly biocompatible, and thusthey should preferably comprise a high proportion of well toleratedcomponents. In one embodiment, for example, the lipid matrix comprisesless than 10% mono-acyl lipids (e.g. glycerol monooleate), because theseare generally less well tolerated than diacyl lipids.

One preferred lipid matrix comprises:

-   -   a) at least one neutral lipid (e.g. diacyl lipid or tocopherol),        preferably at least one diacyl glycerol and/or tocopherol;    -   b) at least one phospholipid;    -   c) at least one oxygenated organic solvent; and    -   d) optionally at least one fragmentation agent.

Such a matrix has a considerable advantage in that the components aretypically very well tolerated by the subject, and furthermore,components and proportions can be selected to provide desirable phasebehaviour.

Such suitable systems are described in detail in, for example,WO2005/117830 and are demonstrated in the examples included in thatpublication, which is incorporated herein by reference. In particular,details and proportions of components (a), (b) and (c) correspond tothose described below and on pages 9 to 17 of WO2005/117830.

In this preferred lipid-based controlled-release matrix, weight ratiosof components a:b may be from 5:95 to 95:5. Preferred ratios wouldgenerally be from 90:10 to 20:80 and more preferably from 85:15 to30:70. The most preferred ratios of a:b are close to parity, especially35:65 to 65:35, more preferably 42:58 to 58:42.

In any embodiment of the invention, and particularly with reference tothe preferred lipid matrix, it is preferable that the formulation is lowviscosity so as to allow ease of administration, and subsequentlyundergoes a phase change following administration. This allows theformulation to become more viscous and/or more bioadhesive afteradministration. Such a phase change may be brought about by a number offactors, but most commonly loss of solvent and/or absorption of water,either or both of which mechanisms may be brought about by exposure toan aqueous fluid.

Thus, the formulations of the invention in all aspects may employ alipid matrix in the form of at least one non-lamellar phase, or maygenerate at least one non-lamellar phase upon exposure to an aqueousfluid. It is preferred that the lipid-based controlled-release matrixforms bulk or particulate ordered phases as described herein.

In all aspects of the present invention, the formulations are preferablylow viscosity mixtures prior to administration. Herein, the term “lowviscosity mixture” is used to indicate a mixture which may be readilyadministered to a subject and in particular readily administered bymeans of a standard syringe and needle arrangement. This may beindicated, for example by the ability to be dispensed from a 1 mldisposable syringe through a19 awg, preferably 22 awg (or a 23 gauge)needle by manual pressure. In a particularly preferred embodiment, thelow viscosity mixture should be a mixture capable of passing through astandard sterile filtration membrane such as a 0.22 μm syringe filter.In other preferred embodiments, a similar functional definition of asuitable viscosity can be defined as the viscosity of a formulation thatcan be sprayed using a compression pump or pressurized spray deviceusing conventional spray equipment. A typical range of suitableviscosities is, for example, 0.1 to 5000 mPas, preferably 1 to 1000 mPasat 20° C.

It has been observed that, by the addition of small amounts of lowviscosity solvent, as indicated herein, a very significant change inviscosity can be provided. For example, in some formulations, theaddition of only 5% of a suitable solvent can reduce viscosity 100-foldand addition of 10% may reduce the viscosity up to 10,000 fold.

Particularly preferred examples of low viscosity mixtures are molecularsolutions and/or isotropic phases such as L2 and/or L3 phases. Asdescribe above, the L3 is a non-lamellar phase of interconnected sheetswhich has some phase structure but lacks the long-range order of aliquid crystalline phase. Unlike liquid crystalline phases, which aregenerally highly viscous, L3 phases are of lower viscosity. Obviously,mixtures of L3 phase and molecular solution and/or particles of L3 phasesuspended in a bulk molecular solution of one or more components arealso suitable. The L2 phase is the so-called “reversed micellar” phaseor microemulsion. Most preferred low viscosity mixtures are molecularsolutions, L3 phases and mixtures thereof L2 phases are less preferred,except in the case of swollen L₂ phases. The L₂ phase as used hereinthroughout is preferably a “swollen” L₂ phase containing greater than 10wt % of solvent (e.g. component c) having a viscosity reducing effect.This is in contrast to a “concentrated” or “unswollen” L₂ phasecontaining no solvent, or a lesser amount of solvent, or containing asolvent (or mixture) which does not provide the decrease in viscosityassociated with the (typically oxygen-containing), low viscositysolvents specified herein.

Following exposure to an aqueous environment, it is preferable that theformulations of all aspects of the invention generate bulk orparticulate ordered phases. Such phases are generally described hereinas “non-lamellar”. The formation of non-lamellar regions in theamphiphile/water, amphiphile/oil and amphiphile/oil/water phase diagramsis a well known phenomenon. Such phases include liquid crystallinephases such as the cubic P, cubic D, cubic G and hexagonal phases, whichare fluid at the molecular level but show significant long-range order,and the L3 phase which comprises a multiply interconnected bi-continuousnetwork of bilayer sheets which are non-lamellar but lack the long-rangeorder of the liquid crystalline phases. Depending upon their curvatureof the amphiphile sheets, these phases may be described as normal (meancurvature towards the apolar region) or reversed (mean curvature towardsthe polar region).

The non-lamellar liquid crystalline and L₃ phases are thermodynamicallystable systems. That is to say, they are not simply a meta-stable statethat will separate and/or reform into layers, lamellar phases or thelike, but are the stable thermodynamic form of the lipid/solventmixture. Bulk liquid crystalline phases are highly viscous and areadvantageous for the formation of depot compositions wherein controlledrelease is desired over a prolonged period, especially followingparenteral administration. L₃ and L₂ phases and dispersed particles ofnon-lamellar phases are typically lower viscosity and more suited tocontrolled release over shorter time periods, as well as to topicalrelease at body surfaces, both internal and external.

In one preferred embodiment of the invention, the formulations form abulk non-lamellar phase upon exposure to an aqueous fluid, particularlya body fluid. This typically occurs in vivo. Bulk non-lamellar phasesare particularly suitable for forming long acting formulations, whichact as a “depot” of active agent, potentially releasing this over a longperiod. This period may be controlled by adding relatively smallquantities of the fragmentation agents (such as polysorbate 80) asdescribed herein. 0 to 5% of fragmentation agent is typical for a bulknon-lamellar phase.

In an alternative embodiment, the formulations generate particles (suchas colloidal) particles spontaneously upon exposure to an aqueous fluid,particularly a body fluid. This typically occurs in vivo. Fragmentingcompositions may be used to generate shorter-acting depot compositions,or may be used over shorter periods to control the more rapid deliveryof an active agent. Fragmenting or particulate non-lamellar phases aretypically generated from compositions comprising a higher proportion offragmentation agent (such as polysorbate 80), as described herein. 5 to30% of fragmentation agent is typical for particulate non-lamellarphases. In all embodiments, all fragmentation agents discussed hereinare suitable.

Component “(a)” as indicated herein is a neutral lipid componentcomprising a polar “head” group and also non-polar “tail” groups.Generally the head and tail portions of the lipid will be joined by anester moiety but this attachment may be by means of an ether, an amide,a carbon-carbon bond or other attachment. Preferred polar head groupsare non-ionic and include polyols such as glycerol, diglycerol and sugarmoieties (such as inositol and glucosyl based moieties); and esters ofpolyols, such as acetate or succinate esters. Preferred polar groups areglycerol and diglycerol, especially glycerol.

In one preferred aspect, component (a) is a diacyl lipid in that it hastwo non-polar “tail” groups. This is generally preferable to the use ofmono-acyl (“lyso”) lipids because these are typically less welltolerated in vivo. The two non-polar groups may have the same or adiffering number of carbon atoms and may each independently be saturatedor unsaturated. Examples of non-polar groups include C₆-C₃₂ alkyl andalkenyl groups, which are typically present as the esters of long chaincarboxylic acids. These are often described by reference to the numberof carbon atoms and the number of unsaturations in the carbon chain.Thus, CX:Z indicates a hydrocarbon chain having X carbon atoms and Zunsaturations. Examples particularly include caproyl (C6:0), capryloyl(C8:0), capryl (C10:0), lauroyl (C12:0), myristoyl (C14:0), palmitoyl(C16:0), phytanoly (C16:0), palmitoleoyl (C16:1), stearoyl (C18:0),oleoyl (C18:1), elaidoyl (C18:1), linoleoyl (C18:2), linolenoyl (C18:3),arachidonoyl (C20:4), behenoyl (C22:0) and lignoceroyl (C24:9) groups.Thus, typical non-polar chains are based on the fatty acids of naturalester lipids, including caproic, caprylic, capric, lauric, myristic,palmitic, phytanic, palmitolic, stearic, oleic, elaidic, linoleic,linolenic, arachidonic, behenic or lignoceric acids, or thecorresponding alcohols. Preferable non-polar chains are palmitic,stearic, oleic and linoleic acids, particularly oleic acid.

The diacyl lipid, when used as all or part of component (a), may besynthetic or may be derived from a purified and/or chemically modifiednatural sources such as vegetable oils. Mixtures of any number of diacyllipids may be used as component (a). Most preferably this component willinclude at least a portion of diacyl glycerol (DAG), especially glyceroldioleate (GDO). In one favoured embodiment, component (a) consists of orconsists essentially of DAGs. These may be a single DAG or a mixture ofDAGs. A highly preferred example is DAG comprising at least 50%,preferably at least 80% and even comprising substantially 100% GDO.

An alternative or additional highly preferred class of compounds for useas all or part of component (a) are tocopherols. As used herein, theterm “a tocopherol” is used to indicate the non-ionic lipid tocopherol,often known as vitamin E, and/or any suitable salts and/or analoguesthereof. Suitable analogues will be those providing the phase-behaviour,lack of toxicity, and phase change upon exposure to aqueous fluids,which characterise the compositions of the present invention. Suchanalogues will generally not form liquid crystalline phase structures asa pure compound in water. The most preferred of the tocopherols istocopherol itself, having the structure below. Evidently, particularlywhere this is purified from a natural source, there may be a smallproportion of non-tocopherol “contaminant” but this will not besufficient to alter the advantageous phase-behaviour or lack oftoxicity. Typically, a tocopherol will contain no more than 10% ofnon-tocopherol-analogue compounds, preferably no more than 5% and mostpreferably no more than 2% by weight.

In a further advantageous embodiment of the invention, component (a)consists essentially of tocopherols, in particular tocopherol as shownabove.

A preferred combination of constituents for component (a) is a mixtureof at least one DAG (e.g. GDO) with at least one tocopherol. Suchmixtures include 2:98 to 98:2 by weight tocopherol:GDO, e.g.10:90 to90:10 tocopherol:GDO and especially 20:80 to 80:20 of these compounds.Similar mixtures of tocopherol with other DAGs are also suitable.

Component “(b)” in the present invention is at least one phospholipid.As with component (a), this component comprises a polar head group andat least one non-polar tail group. The difference between components (a)and (b) lies principally in the polar group. The non-polar portions maythus suitably be derived from the fatty acids or corresponding alcoholsconsidered above for component (a). It will typically be the case thatthe phospholipid will contain two non-polar groups, although one or moreconstituents of this component may have one non-polar moiety. Where morethan one non-polar group is present these may be the same or different.

Preferred phospholipid polar “head” groups include phosphocholine,phosphoethanolamine, phosphoserine and phosphoinositol. Most preferredis phosphocholine, making phosphatidyl choline (PC) the preferredconstituent of component (b). In a preferred embodiment, component (b)thus consists of at least 50% PC, preferably at least 70% PC and mostpreferably at least 80% PC. Component (b) may consist essentially of PC.

The phospholipid portion, even more suitably than any diacyl lipidportion, may be derived from a natural source. Suitable sources ofphospholipids include egg, heart (e.g. bovine), brain, liver (e.g.bovine) and plant sources including soybean. Such sources may provideone or more constituents of component (b), which may comprise anymixture of phospholipids.

Because the formulations of the invention are to be administered to asubject for the controlled release of an active agent, it is preferablethat the components (a) and (b) are biocompatible. In this regard, it ispreferable to use, for example, diacyl lipids and phospholipids ratherthan mono-acyl (lyso) compounds. A notable exception to this istocopherol, as described above. Although having only one alkyl chain,this is not a “lyso” lipid in the conventional sense and is notencompassed by “lyso” lipids, or “monoacyl” lipids as used herein. Thenature of tocopherol as a well tolerated essential vitamin makes ithighly biocompatible.

Two particularly preferred combinations of components (a) and (b) areGDO with PC and tocopherol with PC, especially in the region 30-90wt %GDO/tocopherol, 10-60 wt % PC and 1-30% solvent (especially ethanol,benzyl alcohol, n-methyl pyrrolidone (NMP) and/or isopropanol).

In addition to amphiphilic components (a) and (b), the preferred lipidmatrices of the invention may also contain additional amphiphiliccomponents, although it is preferred that these are at relatively lowlevels. In one embodiment of the invention, the pre-formulation containsup to 10% (by weight of components (a) and (b)) of a charged amphiphile,particularly an anionic amphiphile such as a fatty acid or anionicphospholipid. Preferred fatty acids for this purpose include caproic,caprylic, capric, lauric, myristic, palmitic, phytanic, palmitolic,stearic, oleic, elaidic, linoleic, linolenic, arachidonic, behenic orlignoceric acids, or the corresponding alcohols. Preferable fatty acidsare palmitic, stearic, oleic and linoleic acids, particularly oleicacid. Preferred anionic phospholipids include phosphatidylglycerol (PG),phosphatidylserine (PS) and phosphatidic acid (PA). Preferablephospholipids are dioleoylphosphatidylglycerol (DOPG),palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylserine(DOPS), dioleoylphosphatidic acid (DOPA), soy-derivedphosphatidylglycerol and egg-derived phosphatidylglycerol, particularlyDOPG and POPG. It is particularly advantageous that these components beused in combination with a cationic peptide active agent (see below).The combination of an anionic lipid and a cationic peptide is believedto provide a sustained release composition of particular value. This mayin part be due to increased protection of the peptide from thedegradative enzymes present in vivo.

Optional but preferable component “(c)” of the lipid-basedcontrolled-release matrix is an oxygen containing organic solvent.Because the formulations are for use in contact with an aqueous fluid,and particularly a body-fluid (e.g. in vivo), it is desirable that thissolvent be tolerable to the subject and be capable of mixing with theaqueous fluid, and/or diffusing or dissolving out of the pre-formulationinto the aqueous fluid. Solvents having at least moderate watersolubility are thus preferred.

Typical solvents suitable for use as component (c) include at least onesolvent selected from alcohols, ketones, esters (including lactones),ethers, amides and sulphoxides. Examples of suitable alcohols includeethanol, isopropanol, benzyl alcohol and glycerol formal. Monools arepreferred to diols and polyols. Where diols or polyols are used, this ispreferably in combination with an at least equal amount of monool orother preferred solvent. Examples of ketones include acetone, andpropylene carbonate. Suitable ethers include diethylether, glycofurol,diethylene glycol monoethyl ether, dimethylisobarbide, and polyethyleneglycols. Suitable esters include ethyl acetate, benzyl benzoate andisopropyl acetate and dimethyl sulphide is as suitable sulphide solvent.Suitable amides include NMP, 2-pyrrolidone, and dimethylacetamide (DMA),and sulphoxides include dimethylsulphoxide (DMSO). Less preferredsolvents include dimethyl isosorbide, tetrahydrofurfuryl alcohol,diglyme and ethyl lactate.

The solvent component (c) will generally be at least partially lost uponin vivo formation of the depot composition, will evaporate, or will bediluted by absorption of water from the surrounding air and/or tissue.It is preferable, therefore, that component (c) be at least to someextent water miscible or dispersible, and at least should not repelwater to the extent that water absorption is prevented. In this respectalso, oxygen containing solvents with relatively small numbers of carbonatoms (for example up to 10 carbons, preferably up to 8 carbons) arepreferred. Obviously, where more oxygens are present a solvent will tendto remain soluble in water with a larger number of carbon atoms. Thecarbon to heteroatom (e.g. N, O, preferably oxygen) ratio will thusoften be around 1:1 to 6:1, preferably 2:1 to 4:1. Where a solvent witha ratio outside one of these preferred ranges is used then this willpreferably be no more than 75%, preferably no more than 50%, incombination with a preferred solvent (such as ethanol). This may beused, for example, to decrease the rate of evaporation of the solventfrom the pre-formulation to control the rate of liquid crystalline depotformation.

The amount of component (c), where present in the formulations of theinvention and in the lipid-based controlled-release matrix will be atleast sufficient to provide a low viscosity mixture (e.g. a molecularsolution, see above) of all components, and will be easily determinedfor any particular combination of components by standard methods in viewof the present disclosure. The phase behaviour itself may be analysed bytechniques such as visual observation in combination with polarizedlight microscopy, nuclear magnetic resonance, and cryo-transmissionelectron microscopy (cryo-TEM) to look for solutions, L2 or L3 phases,or liquid crystalline phases. Viscosity may be measured directly bystandard means. As described above, an appropriate practical viscosityis that which can effectively be syringed and particularly sterilefiltered. This will be assessed easily as indicated herein. The maximumamount of component (c) to be included will depend upon the exactapplication of the formulation but generally the desired properties willbe provided by any amount forming a low viscosity mixture (e.g. amolecular solution, see above) and/or a solution with sufficiently lowviscosity. Because the administration of unnecessarily large amounts ofsolvent to a subject is generally undesirable the amount of component(c) will typically be limited to no more than ten times (e.g. threetimes) the minimum amount required to form a low viscosity mixture,preferably no more than five times and most preferably no more thantwice this amount. The composition of the present invention may,however, contain a greater quantity of solvent than would be acceptablein an immediate dosage composition. This is because the process by whichthe active agents are slowly released (e.g. formation of shells ofliquid crystalline phase as described herein) also serve to retard thepassage of solvent from the composition. As a result, the solvent isreleased over some time (e.g. minutes or hours) rather thaninstantaneously and so can be better tolerated by the body.

Because viscosity is a highly significant factor in administeringcompositions by injection or spraying, it is preferred that the solventbe itself of very low viscosity. The viscosity of the “low viscosity”solvent component (c) (single solvent or mixture) should typically be nomore than 18 mPas at 20° C. This is preferably no more than 15 mPas,more preferably no more than 10 mPas and most preferably no more than 7mPas at 20° C. Furthermore, the solvent should be suitable for loweringthe viscosity of the matrix, and any other components (e.g. activeagent) in the mixture. Ethanol is particularly preferred as suitable inall of these respects.

Higher proportions of solvent may also be used for non-parenteral (e.g.topical) applications, especially to body surfaces, where the solventwill be lost by evaporation rather than absorbed into the body. For suchapplications up to 100 times the minimum amount of solvent may be used(e.g. up to 95% by weight of the composition, preferably up to 80% byweight and more preferably up to 50% by weight), especially where a verythin layer of the resulting non-parenteral composition is desired.

As a general guide, the weight of component c will typically be around0.5 to 50% of the total weight of the (a)-(b)-(c) (and (d) wherepresent) solution. This proportion is preferably (especially forinjectable compositions) 2 to 30% and more preferably 5 to 20% byweight.

The formulations of the present invention typically do not containsignificant amounts of water. Because it is essentially impossible toremove every trace of water from a lipid composition, this is to betaken as indicating that only such minimal trace of water exists ascannot readily be removed. Such an amount will generally be less than 1%by weight, preferably less that 0.5% by the weight of thepre-formulation. In one preferred aspect, the formulations of theinvention do not contain glycerol, ethylene glycol or propylene glycoland contain no more than a trace of water, as just described.Alternatively, propylene glycol may be present as the sole solvent, oras one component of the solvent.

There is a certain embodiment of the present invention in which higherproportions of water may be tolerated. This is where water is present asa part of the solvent component in combination with an additionalwater-miscible component (c) (single solvent or mixture). In thisembodiment, up to 20%, preferably up to 10 wt % water may be presentproviding that at least 3 wt %, preferably at least 5% and morepreferably at least 7 wt % component (c) is also present, that component(c) is water miscible, and that the resulting pre-formulation remainsnon-viscous and thus does not form a liquid crystalline phase. Generallythe weight ratio between organic solvent component (c) and water will bebetween 20:80 and 80:20, preferably 30:70 to 70:30 and more preferably35:65 to 65:35. In one embodiment the proportion is at least 50%solvent. Most suitable solvents of use with water in this aspect of theinvention include ethanol, isopropyl alcohol, NMP, acetone and ethylacetate.

As optional but preferable fragmentation agent component (d) canfunction any amphiphile capable of serving as a fragmentation agent withthe selected components (a) and (b) (and (c), where present). Afragmentation agent is a (pure or mixed) agent which allows thecomposition comprising components (a) and (b) to form (byself-dispersion or by the input of energy, such as by shearing orsonication) structured particles, as described herein. Particularlysuitable particles are e.g. non-lamellar, especially liquid crystalline,L₂ or L₃. Non lamellar phases are described in greater detail hereinabove.

There are a number of different molecular classes that are suitable asfragmentation agents in the present invention. These include;

1) Polymeric agents: Poloxamers (preferably Pluronic® F127, Pluronic®F68, Pluronic® F108 Pluronic® L44), 2-Methacryloyloxyethylphosphorylcholine n-butyl methacrylate co-block polymers (such asPUREBRIGHT MB-37-50T and PUREBRIGHT MB-37-100T from NOF Corp.),pegylated sorbitan fatty acid esters (polysorbates, particularlyPolysorbate 80), PEGylated surfactants (e.g. Solutol HS15 from BASF),pegylated castor oil derivatives (e.g. Cremophor EL, Cremophor RH40),pegylated fatty acids (e.g. PEG-oleate), pegylated phospholipids(including DOPE-PEG(2000), DOPE-PEG(5000) and DSPE-PEG(5000)),polyglycerin(PG)-phospholipids (such as DSPE-PG, for example, SUNBRIGHTDSPE-PG8G from NOF Corp., DOPE-PG), pegylated oligoalkylsorbitols (suchas PEG-60 Sorbitoltetraoleate, e.g. GO-460V from Nikko Chemicals),pegylated glyceryl fatty acid esters (e.g. TMGO-15 (Nikko Chemicals)),pegylated tocopherols such as d- alpha tocopheryl polyethylene glycol1000 succinate (Vitamin E TPGS (Eastman)) and pegylated alkyl ethers;

2) Polyol surfactants: sugar derived alkyl esters (such as sucroselaurate and sucrose oleate), sugar derived alkyl ethers (e.g. octylglucoside);

3) Proteins: including casein, sodium caseinate, lysozyme;

4) Anionic surfactants: Carboxylates of fatty acids (especially sodiumoleate, sodium palmitate, sodium stearate, sodium myristate), alkylsulfates (such as sodium dodecyl sulphate (SDS)); and

5) Cationic surfactants: alkyl ammonium salts (including dodecyltrimethyl ammonium bromide (DTAB), cetyl trimethyl ammonium bromide(CTAB) and oleyl ammonium chloride).

Generally, in the present invention, protein fragmentation agents, suchas those described in (3) above are less preferred. Class (1) asdescribed above also includes fragmentation agents that are highlysuitable for this purpose.

The majority of the (d)-components form normal micellar (L1) phases oncontact with excess water. However, the components need not formmicelles to function as fragmentation agents. The effective functioningof a fragmentation agent will easily be tested by a skilled worker bypreparing appropriate compositions and conducting simple tests asillustrated in the Examples herein, and also by reference toWO2006/013369 (particularly the Examples), the disclosure of which isincorporated herein by reference.

Where component (d) is present, the components (a), (b) and (d) willtypically be present in the following proportions (where a, b and d arethe weights of components (a), (b) and (d) respectively); d/(a+b+d) isbetween 0.01 and 0.3. Compositions within this range have a hightendency to self-disperse or to form stable particles followingdispersion with or without energy input. It is preferred, especiallywhere it is desired to provide self-dispersion and greatest particlesize control that the proportions of (a), (b) and (d) are such thata/(a+b+d) is between 0.25 (e.g. 0.35) and 0.80 (e.g. 0.75), morepreferably 0.35 (e.g. 0.4) and 0.75 (e.g. 0.65) and d/(a+b+d) is between0.03 and 0.25 (e.g. 0.2) (where a, b and d are the weights of components(a), (b) and (d) respectively).

In one embodiment, the surfactant is one of types 1, 3, 4 or 5, shownabove, most preferably a polymer surfactant.

As with all components indicated explicitly or implicitly herein asoptional, components (c) and (d) may each independently be present orabsent.

One of the key components of the present invention is the thiolatedantioxidant. Like essentially all organic molecules, lipids andbiologically active agents are thermodynamically unstable to oxidation.As a result, many lipid formulations, including those comprisingbioactive agents such as APIs are susceptible to degradation uponstorage, especially by oxidation.

Unfortunately, many common antioxidants are not highly compatible withlipid systems. Indeed, the present inventors have surprisinglyestablished that some antioxidants commonly used in previous systems cancause increased degradation of active agents in a lipid system. Thisapplies particularly to peptide active agents. The present inventorshave therefore analysed a variety of potential antioxidant compounds andclasses for use with lipid based matrix systems and have surprisinglyfound that one particularly class of antioxidants is unusually wellsuited for use in these systems.

The present inventors have now established that thiolated antioxidants,particularly mono-thioglycerol (MTG) and cysteine analogues such asN-acetyl cysteine, are highly effective in lipid based systems, and thusin the present invention the antioxidant component is a thiolatedantioxidant, preferably thiolated sugar, thiolated amino acid, athiolated amino ester, or a thiolated polyol. Mono-thioglycerol,N-acetyl cysteine or cysteine are preferred thiolated antioxidants.

The antioxidant component is generally included in the range 0.01 to2.0% by weight of the total composition (formulation). This is mostpreferably 0.05 to 1.0%, and around 0.2 to 0.5% of antioxidant(particularly MTG) is particularly preferred, especially in combinationwith the other preferred components and ranges indicated herein aboveand below.

The reason for the utility of thiolated antioxidants in general and MTGin particular is not known. Without being bound by theory, it isbelieved that MTG acts as an effective chain-breaking donatingantioxidant according to established mechanisms whereby peroxyl radicals(ROO.) are neutralized. Their quenching by the thiolated antioxidantbreaks the cycle of further oxidative degradation. Thiols such as MTGand N-acetyl cysteine may also regenerate certain components from theiroxidized forms.

Stability data using a number of different antioxidants shown in theExamples below demonstrate that thiolated antioxidants are surprisinglymore efficient than other antioxidants in suppressing the oxidativedegradation of bioactive agents. This effect is outlined in the Examplesand tables below and in the attached Figures.

The present inventors have additionally established that the combinationof a thiolated antioxidant compound and a chelating agent provides ahighly effective combination in stabilising the lipid based compositionsof the invention. In all aspects of the invention, the antioxidantcomponent may thus be supplemented with a chelator. Suitable chelatingagents include any poly-dentate (including bi-dentate) ligand, includingpoly-acids, poly-amides, poly-amines and poly-ethers. Manymetal-chelating ligands are known to those skilled in the art, and willbe suitable for use in the present invention. Preferred chelating agentsinclude diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA) and the corresponding sodium, disodium andcalcium disodium salts of EDTA. Citric acid, and its biologicallytolerable salts (e.g. sodium, calcium, potassium and/or magnesium salts)also functions as a chelator and could be used as the chelating agent inthe method of the present invention. The amounts of chelator requiredmay be quite small, and thus citric acid may act as a chelator atconcentrations equal to or below that required to have alipid-soluble-acid effect (see below). In an alternative embodiment,citric acid may be excluded from the pre-formulations of the invention.

Without being bound by theory, it is thought that the metal chelatingnature of the chelating agent serves to compliment the chain-breakingdonating antioxidant property of the thiolated antioxidant, thus servingtogether to reduce the formation of oxygenated radical species, andquench those that do form.

The chelating agent is generally included in the range 0.005 to 1.0% byweight of the total composition (formulation). This is most preferably0.01 to 0.8%, and around 0.02 to 0.5% of chelating agent is particularlypreferred, especially in combination with the other preferred componentsand ranges indicated herein above.

One useful embodiment of the present invention combines an antioxidant,as described herein, a chelating agent, and a lipid composition whichforms particles of non-lamellar phase upon exposure to a body fluid.Such compositions typically comprise a fragmentation agent such aspolysorbate 80 (P80) and typical lipid components are described herein.MTG, EDTA, GDO, PC and P80 form a highly preferred combination, alongwith an optional organic solvent such as ethanol and/or propyleneglycol. The addition of citric acid, or use of citric acid in place ofEDTA is also a valuable embodiment. The amounts of each component whichare suitable are those discussed herein.

An optional component of the formulations of the present invention is abioactive agent. In one embodiment, the lipid matrices stabilised bythiolated antioxidant may be used without any additional bioactiveagent, and in particular without any active pharmaceutical agent (API),for example for their soothing and/or protective properties atbiological surfaces such as mucosal surfaces. In an alternative andpreferred embodiment, however, one or more bioactive agent is includedin the formulations.

As used herein, the term “bioactive agent” (described equivalently as“active agent” herein) may be any compound having a desired biologicalor physiological effect, such as a protein, drug, antigen, nutrient,cosmetic, fragrance, flavouring, diagnostic, pharmaceutical, vitamin, ordietary agent and will be formulated at a level sufficient to provide anin vivo concentration at a functional level (including localconcentrations for topical compositions). Under some circumstances oneor more of components of the lipid matrix i) (e.g. components (a), (b),(c) and/or (d)) may also be an active agent, although it is preferredthat the optional bioactive agent (iii) should not be one of thesecomponents (e.g. should not be a component of the lipid matrix). Mostpreferred active agents are pharmaceutical agents (e.g. APIs) includingdrugs, vaccines, and diagnostic agents.

Drug agents that may be delivered by the present invention andformulated therewith include drugs which act on cells and receptors,peripheral nerves, adrenergic receptors, cholinergic receptors, theskeletal muscles, the cardiovascular system, smooth muscles, the bloodcirculation system, endocrine and hormone system, blood circulatorysystem, synoptic sites, neuroeffector junctional sites, theimmunological system, the reproductive system, the skeletal system,autacoid system, the alimentary and excretory systems, the histaminesystem, and the central nervous system.

Examples of drugs which may be formulated in the compositions of thepresent invention include, but are not limited to, antibacterial agentssuch as (3-lactams or macrocyclic peptide antibiotics, anti fungalagents such as polyene macrolides (e.g. amphotericin B) or azoleantifungals, anticancer and/or anti viral drugs such as nucleosideanalogues, paclitaxel and derivatives thereof, docetaxel and derivativesthereof, anti inflammatorys, such as non-steroidal anti inflammatorydrugs and corticosteroids, cardiovascular drugs including cholesterollowering and blood-pressure lowing agents, analgesics, antipsychoticsand antidepressants including seritonin uptake inhibitors,prostaglandins and derivatives, vaccines, and bone modulators.Diagnostic agents include radionuclide labelled compounds and contrastagents including X-ray, ultrasound and MRI contrast enhancing agents.Nutrients include vitamins, coenzymes, dietary supplements, etc.

Particularly suitable active agents include those which would normallyhave a short residence time in the body due to rapid breakdown orexcretion, those with poor oral bioavailability and particularly thosewhich are susceptible to oxidative degradation. These include peptide,protein and nucleic acid based active agents, hormones and othernaturally occurring agents in their native or modified forms. In onehighly preferred embodiment of the present invention, such agents areadministered in the form of a lipid depot composition formed, forexample, from the preferred lipid matrices described herein. In thisway, the active agents are provided at a sustained level for a length oftime which may stretch to days, weeks or even several months in spite ofhaving rapid clearance rates, and can be kept at a desired dosage levelfor an extended period due to effective protection from oxidativedegradation. This offers obvious advantages in terms of dosage stabilityand patient compliance over dosing multiple times each day for the sameperiod. In one preferred embodiment, the active agent thus has abiological half life (upon entry into the blood stream) of less than 1day, preferably less than 12 hours and more preferably less than 6hours. In some cases this may be as low as 1-3 hours or less. Suitableagents are also those with poor oral bioavailability relative to thatachieved by injection, for where the active agent also or alternativelyhas a bioavailability of below 0.1%, especially below 0.05% in oralformulations.

Peptide and protein based active agents include human and veterinarydrugs selected from the group consisting of adrenocorticotropic hormone(ACTH) and its fragments, angiotensin and its related peptides,antibodies and their fragments, antigens and their fragments, atrialnatriuretic peptides, bioadhesive peptides, Bradykinins and theirrelated peptides, calcitonins and their related peptides, cell surfacereceptor protein fragments, chemotactic peptides, cyclosporins,cytokines, Dynorphins and their related peptides, endorphins andP-lidotropin fragments, enkephalin and their related proteins, enzymeinhibitors, immunostimulating peptides and polyaminoacids, fibronectinfragments and their related peptides, gastrointestinal peptides,gonadotrophin-releasing hormone (GnRH) agonists and antagonist,glucagon, glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) (plus otherpeptide GLP-1 and GLP-2 receptor agonists), growth hormone releasingpeptides, immunostimulating peptides, insulins and insulin-like growthfactors, interleukins, luthenizing hormone releasing hormones (LHRH) andtheir related peptides, melanocyte stimulating hormones and theirrelated peptides, nuclear localization signal related peptides,neurotensins and their related peptides, neurotransmitter peptides,opioid peptides, oxytocins, vasopressins and their related peptides,parathyroid hormone and its fragments, protein kinases and their relatedpeptides, somatostatins and their related peptides, substance P and itsrelated peptides, transforming growth factors (TGF) and their relatedpeptides, tumor necrosis factor fragments, toxins and toxoids andfunctional peptides such as anticancer peptides including angiostatins,antihypertension peptides, anti-blood clotting peptides, andantimicrobial peptides; selected from the group consisting of proteinssuch as immunoglobulins, angiogenins, bone morphogenic proteins,chemokines, colony stimulating factors (CSF), cytokines, growth factors,interferons (Type I and II), interleukins, leptins, leukaemia inhibitoryfactors, stem cell factors, transforming growth factors and tumornecrosis factors.

A further considerable advantage of the depot compositions of thepresent invention is that active agents are released gradually over longperiods without the need for repeated dosing. The compositions are thushighly suitable for situations where patient compliance is difficult,unreliable or where a level dosage is highly important, such asmood-altering actives, those actives with a narrow therapeutic window,and those administered to children or to people who's lifestyle isincompatible with a reliable dosing regime. The compositions of theinvention are also useful for “lifestyle” APIs where the inconvenienceof repeated dosing might outweigh the benefit of the API. Particularclasses of APIs for which this aspect offers a particular advantageinclude contraceptives, hormones (including contraceptive hormones andparticularly hormones used in children such as growth hormone),anti-addictive agents, supplements such as vitamin or mineralsupplements, anti-depressants and anticonvulsants.

Cationic peptides are suitable for use, particularly in embodimentswhere a portion of the lipid matrix part of the formulation comprises ananionic amphiphile such as a fatty acid or anionic phospholipid. In thisembodiment, preferred peptides include calcitonin, oxytocin,interferon-beta and -gamma, interleukins 4, 5, 7 and 8 and otherpeptides having an isoelectric point above pH 7, especially above pH 8.

Because the antioxidants used in the present invention are slightlyreducing, it is preferred that the active agents used in the method ofthe invention should not be susceptible to permanent inactivation byreduction. Thus, for example, peptide active agents are preferably thosewhich are not reduction-sensitive. Typically, peptides (includingproteins) which form one or more disulphide linkages may be susceptibleto reductive inactivation, and so are less preferred. In some cases,however, the necessary cross-links re-form spontaneously upon exposureto the normal oxidative environment in vivo, and in such cases thepeptide agents remain suitable for use in the present invention.

Where present, the amount of bioactive agent to be formulated in thepresent invention will depend upon the functional dose and the periodduring which the composition formed upon administration is to providesustained release. Typically, the dose formulated for a particular agentwill be around the equivalent of the normal daily dose multiplied by thenumber of days the formulation is to provide release. This amount can betailored to take into account any adverse effects of a large dose at thebeginning of treatment, as this will generally be the maximum dose used.The precise amount suitable in any case can readily be determined bysuitable experimentation in view of the present disclosure.

In one embodiment, the pre-formulations of the present invention will beadministered parenterally. This administration will generally not be anintra-vascular method but will preferably be subcutaneous intracavitaryor intramuscular or subcutaneous. Typically the administration will beby injection, which term is used herein to indicate any method in whichthe formulation is passed through the skin or mucosal surface, such asby needle, catheter or needle-less injector.

In parenteral (especially subcutaneous) depot precursor formulation,preferred active agents are those suitable for systemic administrationincluding antibacterials (including amicacin, monocyclineanddoxycycline), local and systemic analgesics (including bupivacain,tramadol, fentanyl, morphine, hydromorphone, methadone, buprenorphine,oxycodone, codeine, asperine, acetaminophen), NSAIDS (such asibuprofene, naproxene, keteprofene, indomethansine, sulindac, tolmethin,salysylic acids such as salisylamide, diflunisal), Cox1 or Cox2inhibitors (such as celecoxib, rofecoxib, valdecoxib), anticancer agents(including octreotide, lanreotide, buserelin, luprorelin, goserelin,triptorelin, avorelin, deslorein, abarelix, degarelix, fulvestrant,interferon alpha, interferon beta, darbepoetin alpha, epoetin alpha,beta, delta, docetaxel and paclitaxel), antipsychotics (likebromperidol, risperidone, olanzapine, iloperidone, paliperadone,pipotiazine and zuclopenthixol), antivirals, anticonvulsants (forinstance tiagabine topiramate or gabapentin) or nicotine, hormones (suchas testosterone, and testosterone undecanoate, medroxyprogesterone,estradiol) growth hormones (like human growth hormone), and growthfactors (like granulocyte macrophage colony-stimulating factor). Peptideactive agents such as glucagon and opiate active agents such asBuprenorphine are particularly preferred active agents in allembodiments of the present invention.

In an alternative embodiment, the formulations of the present inventionmay form non-parenteral depots where the active agent is slowly releasedat a body surface. It is especially important in this embodiment thatthe formulations of the invention and/or the (preferably non-lamellar,e.g. liquid crystalline) depot compositions formed therefrom shouldpreferably be bioadhesive. That is to say that the compositions shouldcoat the surface to which they are applied and/or upon which they formas appropriate and should remain even when this surface is subject to aflow of air or liquid and/or when the surface is rubbed. It isparticularly preferable that the depot compositions formed should bestable to rinsing with water. For example, a small volume of depotprecursor may be applied to a body surface and be exposed to a flow offive hundred times its own volume of water per minute for 5 minutes.After this treatment, the composition can be considered bioadhesive ifless than 50% of the bioactive agent has been lost. Preferably thislevel of loss will be matched when water equalling 1000 times and morepreferably 10 000 times the volume of the composition is flowed past perminute for five, or preferably 10, minutes.

Although the non-parenteral compositions of the present invention mayabsorb some or all of the water needed to form a liquid crystallinephase structure from the biological surfaces with which they arecontacted, some additional water may also be absorbed from thesurrounding air. In particular, where a thin layer of high surface areais formed then the affinity of the composition for water may besufficient for it to form a liquid crystalline phase structure bycontact with the water in the air. The “aqueous fluid” referred toherein is thus, at least partially, air containing some moisture in thisembodiment.

Non-parenteral depot compositions will typically be generated byapplying the formulations described herein topically to a body surfaceor to a natural or artificially generated body cavity and/or to thesurface of an implant. This application may be by direct application ofliquid such as by spraying, dipping, rinsing, application from a pad orball roller, intra-cavity injection (e.g to an open cavity with orwithout the use of a needle), painting, dropping (especially into theeyes) and similar methods. A highly effective method is aerosol or pumpspraying, and this requires that the viscosity of the pre-formulation beas low as possible and is thus highly suited to the preferred lipidcompositions of the invention. Non-parenteral depots may, however, beused to administer systemic agents e.g. transmucosally or transdermally.

Non-parenteral depots may also be used for application to surfaces,particularly of implants and materials which will be in contact with thebody or a body part or fluid. Devices such as implants, catheters,stents and the like may thus be treated e.g. by dipping or spraying withthe formulations of the invention, which will form a robust layer toreduce the introduction of infection. Anti-infective actives areparticularly suited to this aspect.

A further advantageous component which may be included in theformulations of the present invention is a lipid soluble acid. Theoptional “lipid soluble acid” component is generally a low molecularweight compound which would form an acidic solution in an aqueous medium(i.e. in water). Although referred to as an “acid” herein, and acting asan acid in aqueous solutions, this component does not generally act as atypical acid in the pre-formulations of the invention, because these arelipid-based and thus generally non-aqueous. In one embodiment, such alipid soluble acid has a molecular weight of less than 500 amu, e.g.less than 300 amu and less than 200 amu. Organic and mineral acids areuseful lipid soluble acids for purposes of the present invention,especially those having low molecular weight as indicated. The lipidsoluble acids will generally be those having a pKa of lower than 5, suchas lower than 4.7 and lower than 4.5. The acids must also be suitablefor dissolution at the required level in the chosen matrix system. Asthe matrices are generally hydrophobic or amphiphilic, suitable acidsare referred to herein as “lipid soluble”. Because the lipid solubleacids are often administered as part of a parenteral drug-releasesystem, biocompatibility in the relevant quantities is also necessary.Suitable lipid soluble acids include those selected from citric acid,benzoic acid, sulphonic acids (e.g. methane sulphonic acid, benzenesulphonic acid or toluene sulphonic acid) and hydrohalic acids (e.g.hydrochloric acid, hydrobromic acid or hydoriodic acid). In variousembodiments, lipid soluble acids are citric acid, methane sulphonicacid, benzene sulphonic acid, benzoic acid, toluene sulphonic acid andHCl. Citric acid and benzoic acid are highly preferred and preferablyused in combination with PC (soy and/or DOPC), GDO, ethanol andoptionally PG. This use in combination may be in the component ratios asdescribed herein above.

In one alternative embodiment of the invention, the lipid soluble acidis not a hydrohalic acid (e.g. not HCl, not HBr and/or not HI). In thisembodiment, the lipid soluble acid may be, in some embodiments, benzoicacid, citric acid or a sulphonic acid.

In another alternative embodiment of the invention, the lipid solubleacid is not one or more of acetic acid, or ascorbic acid. In oneembodiment, the acid may be citric acid. In an alternative embodiment,the acid is an acid other than citric acid.

Typically, the acid functionality will be the only or the dominantfunctional group in an organic lipid-soluble acid for use in the presentinvention, and thus, the acid will optionally not be a fatty acid, (e.g.having a carbon chain greater than C₆), an amino acid, or a poly acid,especially a chelating poly-acid such as EDTA. Thus, for example, in oneembodiment the acid may be an organic acid having one or more, buttypically no more than 5 acid groups, often no more than four andusually no more than three acid groups. Thus, mono-, di- or tri-acidsare preferred.

The lipid soluble acids are referred to herein as “acids” and in oneaspect they are formulated as at least essentially consisting of theacid in free acid form. In an alternative aspect, however, the lipidsoluble acid may be the salt of the corresponding acid as describedherein, wherein the counter-ion is a physiologically acceptable ion suchas an alkali-metal or alkaline earth metal cation, an ammonium ion or asubstituted ammonium ion. A mixture of such ions is also suitable. Inone corresponding embodiment, the counter-ion is the cation of theactive agent, such as a peptide active agent (e.g. a glucagon ion), or amixture of ions including the cation of the active agent.

Without being bound by theory, it is believed that the ions of the lipidsoluble acids serve to stabilise the active agent component. Because thecompositions are typically essentially free of water, the aqueoushydrogen ion concentration, which is the normal basis of pH does notdirectly apply, and the lipid soluble acids must have an additionaleffect in these systems. It is certainly observed that active agentcomponents can be formulated at higher concentrations and/or can be morestable in the compositions of the present invention in the presence ofthe acid component. In this context, stability is the physical stabilityof the compositions as well as the chemical stability of the activeagent.

In some embodiments, the lipid soluble acid can be highly beneficial incombination with a thiolated antioxidant for stabilising an activeagent.

In all aspects of the invention, the lipid soluble acid, where present,is present at a molar ratio of active agent to lipid soluble acid of 1:1to 1:5000, for example 1:100 to 1:1000, preferably 1:100 to 1:300 orfrom 1:1 to 1:30, preferably 1:1 to 1:20, and most preferably 1:2 to1:10. Because typical lipid soluble acids are of lower molecular weightthan typical peptide active agents, the proportion by weight of lipidsoluble acid may be relatively small. For example, with a smallmolecular weight pH adjuster (e.g. less than 500 amu), 0.01 to 5% of thecomposition may be lipid soluble acid, such as 0.05 to 2%. Either theabsence of acid component, or the presence of citric acid at 0.2 to 2%by weight of the complete formulation is typical.

From a weight point of view, the amount of lipid soluble active agentmay depend upon the molecular weight of the acid and the active agentused, and can be calculated from the molar ratios indicated herein. Forexample, with a peptide active agent of molecular weight 3000-4000 amu,and an acid component of molecular weight 150 to 250 amu, the weight oflipid soluble acid in the formulation may be from around 1:1 to 30:1 ofacid:active agent. This will usually be 1:1 to 20:1, more preferably 5:1to 15:1.

In one embodiment of the present invention, the lipid soluble acid mayact to limit aggregation of the active agent, such as a peptide activeagent. The thiol based antioxidants such as MTG are particularlyeffective in combination with the typical acid, including citric acidand benzoic acid. A synergistic effect may develop from these twocomponents when used as indicated herein. In particular, theanti-oxidising and anti-aggregation effects of the respective componentsmay act synergistically in the preservation of active agent in the lipidvehicle as set out herein. Furthermore, the metal chelating effect of achelating agent such as EDTA may act synergistically with thethiol-based antioxidant and/or the lipid soluble acid to preserve theactive agent and/or lipid by preventing the generation and/orpropagation of radicals.

The resistance to oxidation provided in the formulations of the presentinvention has two primary advantages for practical formulations.Firstly, those compositions formulated to release an active agent overlong periods of time do so more efficiently and effectively if thatactive agent is not oxidised during the release period (e.g. while atleast a part of the dose of active agent remains trapped within thestructure generated when the formulation was administered). Secondly,oxidation resistance is advantageous because the formulations will thenhave greater stability to transport and storage. It is necessary thatany formulation which is to be generated in large quantities, packaged,transported, stored and/or distributed has a certain stable lifetime.This should preferably be at least a month, more preferably at least 3months and most preferably at least 6 months. In many cases, this canonly be achieved by providing the components in a separated form, suchthat they must then be combined shortly or immediately beforeadministration. This procedure can be complex and in any case places anadditional burden on the practitioner or user to prepare thecomposition.

The formulations of the present invention in all aspects are preferablyin ready-to-administer form, and are preferably stable in that form atroom temperature and/or at 4° C. for at least one month, preferably atleast 3 months, more preferably at least 6 months. By stable is meantthat the lipid matrix part and any active agent present should be stableto oxidation under the specified conditions, such that at least 80% ofthe initial level of active bioactive agent content remains in itsactive form following storage for the specified period. Preferably, thiswill be at least 90% and most preferably at least 95% of the bioactiveagent present before storage.

The invention will now be further illustrated by reference to thefollowing non-limiting Examples, and the attached Figures, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the effect of MTG concentration on the protection ofbuprenorphine (BUP) in the lipid formulation of the invention towardsoxidation degradation during stress tests with hydrogen peroxide.

FIG. 2 Shows the recovery of BUP (HPLC) after 1 month at 70° C.(accelerated stability testing) as a function of MTG concentration inthe formulation of the invention.

FIG. 3 Shows the assayed glucagon (GLU) content after storage at 25°C./60% RH for 7 days, plotted as the % of initial (time zero) GLUcontent, with different antioxidants included in the formulations.

FIG. 4 Shows the amount of detected degradation products (% Total Area)in GLU formulations after storage at 25° C./60% RH for 7 days, withdifferent antioxidants included in the formulations.

EXAMPLES Abbreviations used in examples:

Name Abbreviation Supplier Phosphatidylcholine, soy SPC Lipoid, GermanyGlycerol dioleate GDO Danisco, Denmark Ethanol (99.5%) EtOH Kemetyl,Sweden Mono-thioglycerol MTG Fluka, Sweden N-acetyl cysteine N-AcCysSigma-Aldrich, Sweden α-tocopherol TOC DSM, Switzerland Propyl gallatePGall Sigma-Aldrich, Sweden Butyl hydroxytoluene BHT Fluka, Sweden

Example 1 Effects of Different Antioxidants in Lipid Formulations ofBuprenorphine (BUP)

Lipid formulations of BUP also containing antioxidant were prepared inthe following way: First, a liquid lipid stock solution ofSPC/GDO/EtOH/antioxidant (or a reference stock solution withoutantioxidant) in the required proportions were prepared by weighing ofall components into glass vials followed by mixing by end-over-endrotation for about 8 h or until completely homogenous liquids wereobtained. Thereafter, BUP (powder) was added to achieve a nominalconcentration of 7.9 wt % in all cases. The final nominal compositionsof the respective formulations are given in Table 1. The antioxidantswere added in an amount corresponding to common previous use inpharmaceutical products.

TABLE 1 Nominal compositions (wt %) of formulations studied FormulationBUP SPC GDO EtOH MTG PGall TOC BHT A 7.9 41.1 41.1 10.0 — — — — B 7.940.6 40.6 9.9 1.1 — — — C 7.9 41.0 41.0 10.0 — 0.03 — — D 7.9 40.9 40.910.0 — — 0.3 — E 7.9 41.0 41.0 10.0 — — — 0.13

The following stress test was performed to evaluate the effect of thefour different antioxidants (Table 1): Oxidative degradation was inducedby adding 20 μL H₂O₂ (30%)/mL formulation followed by equilibration ofthe formulations for 48 hours at RT (dark). Analysis of BUP and anyoxidation degradation product (DP) in the formulations was performed byHPLC (reversed phase column) with UV detection at 288 nm. The relativeretention time (RR) of BUP was set to 1 and the main oxidation DP had anRR of 1.053.

As shown in Table 2, all of the antioxidants had some effect comparedwith the reference formulation, however; MTG was clearly superior inprotecting BUP from oxidation degradation in the lipid formulation.

TABLE 2 Effect of different antioxidants on BUP oxidation in stressedformulations (H₂O₂ treated formulations) Relative area % of mainFormulation Antioxidant oxidation DP (RR = 1.053) A None (referenceformulation) 1.36 B MTG 0.12 C PGall 0.70 D TOC 0.72 E BHT 0.74

Example 2 Effect of Different MTG Concentrations

The protective effect of different MTG concentrations was assessed byperforming a stress test according to Example 1. Briefly, to a lipidformulation of BUP with the nominal composition BUP/SPC/GDO/EtOH=7.9/41.1/41.1/10.0 wt % was added MTG at concentrations between 0-1.0wt %. To the resulting formulations was added 20 μL H₂O₂ (30%)/mLformulation followed by equilibration for 48 hours at RT (dark). Theformulations were thereafter analysed by HPLC as described in Example 1and the relative area % of the main oxidation degradation product (DP)peak (relative retention RR=1.053) was determined. As shown in FIG. 1,already low concentrations of MTG had a significant protective effect.

Example 3 Effect of Different MTG Concentrations on BUP Stability DuringAccelerated Stability Studies

Lipid formulations of BUP (nominal concentration 7.9 wt %) comprisingdifferent concentrations of MTG (0-1.0 wt %) were prepared as describedin Examples 1 and 2. The formulations were filled in glass vials, theheadspace flushed with nitrogen and the vials were capped withTeflon-coated rubber stoppers and tear-off aluminium caps. The vialswere thereafter transferred to a heating cabinet held at 70° C. andstored for 1 month before HPLC analysis as described in Examples 1 and2. As shown in FIG. 2, the addition of low concentrations of MTGincreased the recovery of BUP from the stored samples.

Example 4 Lipid Formulation Comprising N-Acetyl Cysteine (N-AcCys)

A liquid lipid formulation comprising SPC/GDO/EtOH (42.5/42.5/15 wt %)was prepared by weighing all components in a glass vial followed byend-over-end mixing for 4 hours at RT. To the transparent and homogenouslipid formulation was added N-AcCys at a concentration of 0.25 wt %followed by further mixing for 12 hours. The resulting formulation wastransparent and homogenous.

Example 5 Antioxidant Tests

To prevent breakdown of glucagon resulting from oxidation of themethionine residue (giving Met(O)27 glucagon), an antioxidant may beincluded in the compositions of the present invention. To establish themost suitable antioxidant, various common and less common antioxidantswere tested in the peptide/lipid system of the present invention.

The nominal composition of the samples used for the exploratorystability study is given in Table 3. Note that the antioxidant contentin the respective formulations, except for the reference formulations(#229 and #234), was 0.3 wt % for tocopherol (α-TOC), acsorbyl palmitate(AscPalm) and mono-thioglycerol (MTG) and 0.1 wt % for butylhydroxytoluene (BHT). Samples containing MTG were #233 and 235.

TABLE 3 Sample composition of formulations used for the exploratorystability study Formulation Number Composition (wt %) (antioxidantadditive in bold) 229 GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH =0.3/31.2/31.2/3.9/11.8/0.5/20.1/1.0 230GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/α-TOC =0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 231GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/ AscPalm =0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 232GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/BHT =0.3/31.15/31.15/3.85/11.75/0.5/20.1/1.0/0.1 233GLU/SPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/MTG =0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 234GLU/DOPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH =0.3/31.2/31.2/3.9/11.8/0.5/20.1/1.0 235GLU/DOPC/GDO/P80/EtOH/m-Cres/PG/BzCOOH/MTG =0.3/31.1/31.1/3.9/11.7/0.5/20.1/1.0/0.3 GLU = glucagon; SPC = Soyphosphatidylcholine; GDO = Glyceroldioleate; P80 = Polysorbate 80; EtOH= Ethanol; PG = Propylene glycol; m-Cres = meta-Cresol; BzCOOH = Benzoicacid

The stability data using a number of different antioxidants above haveshown that MTG is more efficient than other lipid soluble antioxidantsin suppressing the oxidative degradation of glucagon. This effect isoutlined in the above Table and discussed by reference to the Figuresbelow.

In FIG. 3, the assayed GLU content after storage at 25° C./60% RH for 7days is plotted as the % of initial (time zero) GLU content. It can beseen that MTG gave best oxidation protection, with only this and BHTbeing better than the control (antioxidant-free) composition.a-tocopherol and AscPalm were degrading of glucagon in these tests.

FIG. 4 displays the amount of detected degradation products (% TotalArea) with a relative retention RR<1 and RR>1, respectively, as comparedwith the peak corresponding to GLU. Because the assay is based on anormal phase (NP) HPLC column, degradation products with RR<1 are morehydrophobic compared with GLU whereas more hydrophilic degradationproducts give RR>1.

The major part of the detected degradation products with RR>1 isconstituted by oxidized glucagon (Met(O)27 glucagon). It is clearly seenthat MTG is most efficient in preventing the formation of Met(O)27glucagon.

What is claimed is: 1) A formulation comprising: i) a lipid matrix; ii)at least one thiolated antioxidant; iii) at least one of: <agonadotrophin-releasing hormone (GnRH) agonist; agonadotrophin-releasing hormone (GnRH) antagonist; a luthenizing hormonereleasing hormone (LHRH); and/or a luthenizing hormone releasing hormone(LHRH) related peptide; and iv) optionally at least one chelating agent.2) A formulation as claimed in claim 1, wherein said lipid matrixcomprises: a) at least one diacyl glycerol and/or tocopherol; b) atleast one phospholipid; c) at least one oxygenated organic solvent; andd) optionally at least one fragmentation agent. 3) A formulation asclaimed in claim 2, wherein the lipid matrix is in the form of at leastone non-lamellar phase, or generates at least one non-lamellar phaseupon exposure to an aqueous fluid. 4) A formulation as claimed in claim1, wherein the thiolated antioxidant is selected from the groupconsisting of a thiolated sugar, thiolated amino acid, a thiolated aminoester, and a thiolated polyol. 5) A formulation as claimed in claim 4,wherein said thiolated antioxidant is selected from the group consistingof mono-thioglycerol, cysteine, and N-acetyl cysteine. 6) A formulationas claimed in claim 1 additionally comprising a chelating agent. 7) Aformulation as claimed in claim 6, wherein said chelating agent is EDTA.8) A formulation as claimed in claim 1 additionally comprising a lipidsoluble acid. 9) A formulation as claimed in claim 1, wherein said atleast one gonadotrophin-releasing hormone (GnRH) agonist and/or GnRHantagonist is selected from the group consisting of buserelin,luprorelin, goserelin, triptorelin, avorelin, deslorelin, abarelix, anddegarelix. 10) A method for reducing oxidative degradation in aformulation comprising a lipid matrix and at least one of: agonadotrophin-releasing hormone (GnRH) agonist; agonadotrophin-releasing hormone (GnRH) antagonist; luthenizing hormonereleasing hormone (LHRH); and/or a luthenizing hormone releasing hormone(LHRH) related peptide; said method comprising adding at least onethiolated antioxidant and optionally at least one chelating agent to theformulation. 11) A method according to claim 10, wherein said at leastone gonadotrophin-releasing hormone (GnRH) agonist and/or GnRHantagonist is selected from the group consisting of buserelin,luprorelin, goserelin, triptorelin, avorelin, deslorelin, abarelix, anddegarelix.